Lung disease models on a chip

ABSTRACT

The presently disclosed subject matter provides a biomimetic lung disease model, and methods of its production and use. In one exemplary embodiment, the biomimetic lung disease model can include a first and second microchannel with a membrane coated with airway epithelial cells disposed between the microchannels and at least one device coupled to the biomimetic model that delivers an agent to at least one microchannel. In certain embodiments, the agent is cigarette smoke.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase application under 35 U.S.C. §371 of International Application No. PCT/US2016/044282, filed on Jul.27, 2016, which claims priority to U.S. Provisional Application Ser. No.62/197,444, filed on Jul. 27, 2015, U.S. Provisional Application Ser.No. 62/348,036, filed on Jun. 9, 2016, and U.S. Provisional ApplicationSer. No. 62/348,055, filed on Jun. 9, 2016, all of which areincorporated by reference herein in their entirety.

BACKGROUND

Toxins and pollutants (e.g. cigarette smoke, silica dust, asbestosfibers, grain dust, and bird and animal droppings) are primary causes ofchronic medical conditions and life-threatening malignancies in thelung. Biological underpinnings of these diseases, however, remain poorlyunderstood due to a lack of surrogate models for mechanisticinvestigation of pathological responses to these toxins and pollutantsin a physiological environment.

The structural, functional and environmental complexity of the lung andairways poses certain technical challenges for in vitro investigation ofits physiology and pathology using traditional cell culture models. As aresult, certain research in this area has relied on expensive andtime-consuming ex vivo or in vivo animal studies that can often fail tomodel biological responses in humans. These drawbacks of existing modelscan limit the understanding and the development of new therapeuticapproaches to lung diseases. Therefore, there is a need for a low-cost,human cell-based alternative to current lung disease models.

One approach to meeting these challenges is to leverage microengineeringtechnologies that provide unprecedented capabilities to control cellularmicroenvironment with high spatiotemporal precision and to presentliving cultured cells with external influences and biochemical signalsin a more physiologically relevant context. This has led to thedevelopment of microengineered biomimetic systems such as“organs-on-chips” that simulate complex organ-level physiology. However,there remains a need for additional physiologically relevant, humancell-based alternatives to model lung disease.

SUMMARY

The presently disclosed subject matter provides a biomimetic lung modeland methods of its use. The present disclosure also provides for methodsof fabricating the biomimetic lung model. In an exemplary non-limitingembodiment, the biomimetic lung model can include a body, membrane, alayer of cells, and a device coupled to the body that can deliver anagent to the cells. In certain embodiments, the body can have a firstand second microchannel. In certain embodiments, the first microchannelcan be situated above the second microchannel. In certain embodiments,the membrane can be disposed between the first and second microchannel.In certain embodiments, the membrane can have a first and second side,wherein the first side faces the first microchannel and the second sidefaces the second microchannel. In certain embodiments, the layer ofcells can be disposed on the first side of the membrane. In certainembodiments, the device can deliver an agent to at least onemicrochannel. In certain embodiments, the device can deliver an agent toone of the first or second microchannels. In certain embodiments, thedevice can deliver the agent to the first microchannel.

In certain embodiments, the body can have a single microchannel. Incertain embodiments, a membrane can be disposed at the bottom of thesingle microchannel, with a first side facing the interior of the singlemicrochannel and a second side facing the exterior of the body ormicrochannel. In certain embodiments, the body with a singlemicrochannel and membrane can be placed over a reservoir for feeding.

In certain embodiments, the biomimetic lung model contains most of themajor cellular constituents in the airway niches of the human lung. Incertain embodiments, the layer of cells comprises airway epithelialcells. In certain embodiments, the airway epithelial cells can compriseType I and Type II cells. In certain embodiments, the airway epithelialcells can be from all compartments of the lung, including but notlimited to, nasal epithelial cells, tracheal epithelial cells, bronchialepithelial cells, small airway epithelial cells and/or alveolarepithelial cells, (e.g., Type I and II cells). In certain embodiments,the platform can model all the different segments/depths of the lung. Incertain embodiment, the airway epithelial cells can be from healthyhuman lung. In certain embodiments, the airway epithelial cells can befrom human diseased lung. In certain embodiments, the diseased lung canbe chronically diseased. In certain embodiments, the layer of cells canfurther comprise macrophages. In certain embodiments, the macrophagescan be alveolar, interstitial, intravascular, airway macrophages, and/oran immortalized macrophage cell line (e.g., THP-1). In certainembodiments, a layer of vascular endothelial cells can be attached tothe second side of the membrane.

In certain embodiments, cells from different parts of the lung can becultured in separate devices and then linked together in a serialfashion to mimic the entire respiratory tract.

In certain embodiments, a gel layer can be attached to the second sideof the membrane. In certain embodiments, the gel can compriseextracellular matrix proteins such as, but not limited to, collagen,fibronectin, laminin, hyaluaronic acid, and/or similar materials. Incertain embodiments, the gel can comprise collagen. In certainembodiments, tissue or cells can be embedded in the gel. In certainembodiments, the gel layer allows the embedded cells to communicate withthe layer of cells on the first side of the membrane. In certainembodiments, the cells embedded in the gel layer can be connectivetissue or cells. In certain embodiments, the cells embedded in the gellayer can be basal stromal cells. In certain embodiments, the basalstromal cells can be fibroblasts and/or pericytes. In certainembodiments, the cells embedded in the gel layer can be airway and/orvascular smooth muscle cells. In certain embodiments, the cells embeddedin the gel layer can be extracellular matrix proteins. In certainembodiments, the gel contains tethering materials encased within. Incertain embodiments, hollow tubes and/or self-assembled living vesselcan be created within the gel layer to mimic vascular and lymphaticsupply.

In certain embodiments, the membrane can be a porous material that hasone or more pores with a width from about 0.4 microns to about 10microns. In certain embodiments, the pores have a width from about 0.5microns to about 9 microns, about 0.6 microns to about 8 microns, about0.7 microns to about 7 microns, about 0.8 microns to about 6 microns,about 0.9 microns to about 5 microns, about 1 microns to about 4microns, about 1.5 microns to about 3.5 microns, or about 2 microns toabout 3 microns. In certain embodiments, the membrane can be one of athin clear polyester fiber, a polyester membrane, apolytetrafluoroethylene membrane, an elastomeric (e.g.,poly(dimethylsiloxane) (PDMS), polyurethane) membrane, a paper membrane,an extracellular matrix membrane, or a natural membrane. In certainembodiments, the natural membrane can be collagen, laminin, or acombination of both.

In certain embodiments, the first microchannel can be above the secondmicrochannel. In certain embodiments, the first microchannel replicatesthe dimensions of the airways in the native human lung. In certainembodiments, the first microchannel has a width from about 0.1 mm toabout 2 mm. In certain embodiments, the first microchannel has a widthfrom about 0.5 mm to about 1 mm.

In certain embodiments, the first microchannel has a width from about0.5 mm to about 2 mm. In certain embodiments, the first microchannel hasa width from about 1 mm to about 2 mm. In certain embodiments, the firstmicrochannel has a width from about 0.6 mm to about 1.9 mm, from about0.7 mm to about 1.8 mm, from about 0.8 mm to about 1.7 mm, from about0.9 mm to about 1.6 mm, from about 1 mm to about 1.5 mm, or from about1.2 mm to about 1.4 mm. In certain embodiments, the first microchannelhas a width of at least about 0.5 mm, at least about 0.75 mm, at leastabout 1 mm, at least about 1.25 mm, at least about 1.5 mm, at least able1.75 mm, or at least about 2 mm. In certain embodiments, the firstmicrochannel has a width of about 100 μm to about 500 μm. In certainembodiments, the first microchannel has a width of about 100 μm to about400 μm. In certain embodiments, the first microchannel has a width ofabout 100 μm to about 300 μm. In certain embodiments, the firstmicrochannel has a width of about 100 μm to about 200 μm. In certainembodiments, the first microchannel has a width of about 110 μm to about190 μm, about 120 μm to about 180 μm, about 130 μm to about 170 μm, orabout 140 μm to about 160 μm. In certain embodiments, the firstmicrochannel has a length from about 1000 μm to about 10 mm. In certainembodiments, the first microchannel has a length from about 1010 μm toabout 9 mm, about 1020 μm to about 8 mm, about 1030 μm to about 7 mm,about 1040 μm to about 6 mm, about 1050 μm to about 5 mm, about 1060 μmto about 4 mm, about 1070 μm to about 3 mm, about 1080 μm to about 2 mm,about 1090 μm to about 1900 μm, about 1100 μm to about 1800 μm, about1200 μm to about 1700 μm, about 1300 μm to about 1600 μm, or about 1400μm to about 1500 μm. In certain embodiments, the first microchannel hasa width from about 1 mm to about 2 mm. In certain embodiments, the firstmicrochannel has a length of about 1000 μm. In certain embodiments, thebody includes one or more flow channels. In certain embodiments, thebody further includes one or more microfabricated openings or ports. Incertain embodiments, the biomimetic lung model optimizes air-liquidinterface culture. In certain embodiments, the first microchannel canhave air or gases flowing through the microchannel. In certainembodiments, the first microchannel can have culture medium flowingthrough the microchannel. In certain embodiments, the secondmicrochannel can serve as a reservoir for basal feeding. In certainembodiment, the second microchannel can have culture medium flowingthrough the microchannel. In certain embodiment, the second microchannelcan have cell media held within its reservoir.

In certain embodiments, the device can deliver an agent to the firstmicrochannel. In certain embodiments, the agent can be cigarette smoke,nicotine aerosol, wood smoke, natural plant smoke, silica dust, acrylicdust, particulates, asbestos fibers, solvents, grain dust, birddroppings, and animal droppings. In certain embodiments, the devicedelivers cigarette smoke to the first microchannel. In certainembodiments, the device delivering the cigarette smoke can be anautomatic smoking machine. In certain embodiments, the cigarette smokecan be delivered to the first microchannel such that the distribution ofcigarette smoke mimics cigarette smoke exposure conditions experience bycell linings in the human lung. In certain embodiments, the cigarettesmoke can be more dilute the deeper it moves into the firstmicrochannel.

The presently disclosed subject matter further provides methods forproducing a biomimetic lung model. In certain embodiments, the methodcan include fabricating a body. In certain embodiments, the body canhave first and second microchannel disposed therein. In certainembodiments, the method can include inserting a membrane between thefirst and second microchannels. In certain embodiments, the membrane canhave a first side and a second side. In certain embodiments, the methodcan include adhering a layer of cells to the first side of the membrane.In certain embodiments, the layer of cells comprise airway epithelialcells. In certain embodiments, the method can include integratingmacrophage cells among the airway epithelial cells. In certainembodiments, the method can include coupling at least one device to thebody that delivers an agent to at least one microchannel. In certainembodiments, the method can include delivering an agent to the firstmicrochannel. In certain embodiments, the method can include deliveringan agent to one of the first or second microchannels. In certainembodiments, the agent can be cigarette smoke. In certain embodiments,the method can include delivering a culture medium through the firstand/or second microchannel. In certain embodiments, the method caninclude delivering a culture medium through the first and/or secondmicrochannel and then exchanging the flow of medium to the flow of air(with or without the agent) through the first microchannel.

In certain embodiments, adhering the layer of cells to the first side ofthe membrane can include standard approaches of extracellular matrixcoating of the membrane, for example, but not limited to the use offibronectin, prior to seeding of cells. In certain embodiments, to seedthe cells, a high density cell suspension can be introduced to thechannel and allowed to incubate under static conditions to allow thecells to adhere. In certain embodiments, the cell suspension is allowedto incubate for 2 to 4 hours. In certain embodiments, after the periodof attachment flow can be initiated to allow the washing away ofunattached cells and beginning the perfused culture stage. In certainembodiments, some cell proliferation can occur to fill out the entiremembrane surface. In certain embodiments, cell proliferation is allowedto occur for 2-3 days.

In certain embodiments, the method can include attaching a gel layer tothe second side of the membrane. In certain embodiments, the gel can becomposed of collagen. In certain embodiments, tissue or cells can beembedded in the gel. In certain embodiments, basal stromal tissue orcells can be embedded in the gel.

In certain embodiments, the method can include casting a gel. Gelcasting can involve any standard method known to one of skill in theart. In certain embodiments, techniques are used to induce surfacemodification to promote collegen/ECM anchoring. In certain embodiments,the casting of a gel can include sulfo-sanpah treatment of the membranematerial to promote collagen/ECM anchorage. In certain embodiments, thegel is prepared with cells and pipetted onto the second side of themembrane that has been stamped to a channel. In certain embodiments, thegel is prepared without cells and pipetted onto the second side of themembrane. In certain embodiments, after the gel layer solidifies, theupper channel portion—now with a cast gel under the membrane—can beflipped back over and placed over the reservoir layer to complete thedevice assembly.

In accordance with certain embodiments of the disclosed subject matter,a method of testing the effects of a toxic agent on the layer of cells.In certain embodiments, the method can include providing a biomimeticlung model, as described hereinabove. In certain embodiments, the methodcan include placing an agent of interest in one of the first or secondmicrochannels. In certain embodiments, the method can include simulatingphysiological conditions. In certain embodiments, the method can includemeasuring pathological responses to the agent. In certain embodiments,the method can include measuring tissue hardening in response to theagent. In certain embodiments, the agent of interest can be cigarettesmoke, nicotine aerosol, wood smoke, natural plant smoke, silica dust,acrylic dust, particulates, asbestos fibers, solvents, grain dust, birddroppings, and animal droppings.

In certain embodiments, the biomimetic lung model can be a model of lunginflammatory diseases. For example, the presence of macrophages allowsfor looking at inflammation. In certain embodiments, the biomimetic lungmodel can be a model of cigarette smoke-induced airway disease. Incertain embodiments, the biomimetic lung model can be a model ofsmoke-induced emphysema. In certain embodiments, the biomimetic lungmodel can be a model of chronic obstructive pulmonary disease (COPD). Incertain embodiments, the biomimetic lung model can be a model of lungfibrosis. In certain embodiments, the biomimetic lung model can be amodel of lung cancer and/or a model to examine premalignant changes inepithelial cells exposed to various agents.

The presently disclosed subject matter further provides methods of usingthe disclosed biomimetic lung model. In certain embodiments, thebiomimetic lung model can be used for identifying pharmaceuticalcompositions that can treat or prevent lung disease. In certainembodiments, the biomimetic lung model can be used for identifyingagents harmful to the lung.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B. depicts (A) microengineered biomimetic lung model accordingto certain embodiments (B) microengineered biomimetic lung modelaccording to other certain embodiments.

FIG. 2 depicts a schematic representation of an exemplary modelaccording to the disclosed subject matter.

FIG. 3 depicts a schematic representation of an exemplary methodaccording to the disclosed subject matter.

FIG. 4 . depicts a schematic representation of an exemplary methodaccording to the disclosed subject matter.

FIG. 5 depicts a schematic representation of an exemplary methodaccording to the disclosed subject matter.

FIG. 6 depicts a microengineered biomimetic lung model according tocertain embodiments depicting an exemplary method for delivering culturemedium.

FIG. 7 . depicts a microengineered biomimetic lung model according tocertain embodiments depicting an exemplary method for deliveringcigarette smoke.

FIG. 8 depicts a microengineered biomimetic lung model according tocertain embodiments depicting an exemplary method for deliveringcigarette smoke.

FIG. 9 depicts a schematic of UPR stress response.

FIG. 10 depicts the cellular physiology of the biomimetic model beforethe exposure to an agent.

FIG. 11 depicts UPR induction via the staining of AFT6.

FIG. 12 depicts UPR induction via the staining of phosphorylated EIF2a(pEIF2a).

FIG. 13A-B. depicts UPR induction via staining of AFT6 and pEIF2a in (A)control/air treated cells and (B) smoke exposed cells.

FIG. 14A-B. depicts cellular injury via staining of viable cells withcalcein AM (green) and labeling of dead/dying cells with ethidiumbromide (red) in (A) cells exposed to smoke for 4 hours and (B) cellsexposed to air for 4 hours.

FIG. 15 . depicts cell morphology in cells exposed to either air orsmoke for 12 hours.

FIG. 16A-B. depicts UPR induction via staining of AFT6 and pEIF2a in (A)cells exposed to air for 16 hours and (B) cells exposed to smoke for 16hours.

FIG. 17A-B depicts simulation data of lung models subject to cigarettesmoke particles and vapor.

FIG. 18A-F depicts morphology and viability results of small airwayepithelial cells exposed to smoke of a single cigarette

FIG. 19A-C depicts reduction of UPR activation following initial smokeexposure.

FIG. 20 depicts UPR induction via staining of AFT6 and pEIF2a in COPDcells exposed to smoke.

FIG. 21 depicts the cellular physiology of the biomimetic modelaccording to certain embodiments, wherein the model incorporates the gellayer.

FIG. 22 depicts the cell viability of the biomimetic model after 72hours of incorporating the gel layer.

FIG. 23 depicts the incorporation of macrophages among the airwayepithelial layer.

DETAILED DESCRIPTION

The present disclosure provides a microengineering approach to emulatingand probing lung disease processes (e.g., cigarette smoking-induced) ina tissue-engineered microenvironment that recapitulates the complexityof human airways. The disclosed biomimetic lung model can integratehuman airway epithelial cells, basal stromal tissue, and airway lumenmacrophages with programmable microfluidic delivery of an agent (e.g.,cigarette smoke) to study deleterious effects of its exposure on theairway epithelium. The disclosed biomimetic lung model can also utilizehuman-derived cells to create a microengineered chronic disease modelthat recapitulated constitutive UPR activation typically observed inchronic obstructive pulmonary disease (COPD).

Biomimetic Lung Model

The presently disclosed subject matter provides a biomimetic lung model.For the purpose of illustration and not limitation, FIG. 1 (A and B)provides exemplary biomimetic lung models 100, 200. In certainembodiments, the biomimetic lung model can include a body 10, a membrane20, and a layer of cells 30.

In certain embodiments, the base 10 can include a first 11 and second 12microchannel disposed thereon (FIG. 2 ). In certain embodiments, thesize of the microchannels can replicate the dimensions of the airways inthe native human lung. In certain embodiments the microchannels can beabout 0.1 mm to about 2 mm wide. In certain embodiments themicrochannels can be about 0.1 mm to about 2 mm high. In certainembodiments, the microchannel can be as high as it is wide. In certainembodiments, the height and width of the microchannel can be different.In certain embodiments, the first and second microchannel can have thesame dimensions. In certain embodiments, the first and secondmicrochannel can have the different dimensions. In certain embodiments,the microchannels can be each separately about 0.1 mm to about 2 mm wideand about 0.1 mm to about 2 mm high. In certain embodiments, themicrochannel can be 1 mm×1 mm. In certain embodiments, the microchannelcan be 2 mm×2 mm. In certain embodiments, the microchannel decrease sizeas the airways in the lung do. For example, one end of the microchannelcan be smaller than the other end.

In certain embodiments, one or more of the channels can be about 1000 μmto about 30 mm in length. In certain embodiments, the length is about1000 μm to about 20 mm. In certain embodiments, the length is about 1000μm to about 10 mm. In certain embodiments, the length can be about 2 mmto about 25 mm, about 3 mm to about 20 mm about 4 mm to about 15 mm, orabout about 5 mm to about 10 mm. In certain embodiments, one or more ofthe channels can be about 10 mm in length.

In certain embodiments, the base 10 can include additional channels(e.g., four, six, eight, or more, total channels) in pairs of twodisposed thereon, with each pair having a membrane disposed therebetween(between the first and second outer body portions 13, 14 when portions13, 14 are mounted to one another to form the overall body. In certainembodiments, the base 10 can include channels in sets larger than two(e.g., three, four, or more) such that each of the channels in the setcan be separated from adjacent channels by a membrane. In certainembodiments, the base 10 can include one or more channels that are notadjacent to another channel, or separated from another channel by amembrane. The number of channels and layouts of the channels, includingshape and dimensions, can vary based on the design of the base 10. Incertain embodiments, each channel will have generally similardimensions. In certain embodiments, the channels will have differentdimensions. In certain embodiments, the base and microfluidic channelscan be made of any suitable material, for example and withoutlimitation, glass, metal, alloy, plastic, wood, paper, and polymer.

In certain embodiments, the membrane 20 can be disposed between thefirst 11 and second 12 microchannels such that the first 11 and second12 microchannels can be in fluid communication through the membrane 20.In certain embodiments, the membrane 20 can have a first side 21 and asecond side 22. In certain embodiments, the membrane 20 can be a thinclear polyester membrane and can have about 0.4 microns to about 10microns pores. In certain embodiments, the pores have a diameter fromabout 0.5 microns to about 9 microns, about 0.6 microns to about 8microns, about 0.7 microns to about 7 microns, about 0.8 microns toabout 6 microns, about 0.9 microns to about 5 microns, about 1 micronsto about 4 microns, about 1.5 microns to about 3.5 microns, or about 2microns to about 3 microns. In certain embodiments, the pores can be anysuitable size. In certain embodiments, the pores can have varying poresizes. In certain embodiments, the thickness of the membrane was about 5microns to about 100 microns. In certain embodiments, the thickness ofthe membrane can be about 10 microns to about 90 microns, about 20microns to about 80 microns, about 30 microns to about 70 microns, about40 micros to about 60 microns. In certain embodiments, the thickness ofthe membrane is at least about 5 microns, at least about 10 microns, atleast about 20 microns, at least about 30 microns, at least about 40microns, at least about 50 microns, at least about 60 microns, at leastabout 70 microns, at least about 80 microns, at least about 90 microns,or at least about 100 microns. In certain embodiments, the membrane caninclude porous portions and non-porous portions. In certain embodiments,the membrane 20 can be a polyester membrane, a polytetrafluoroethylenemembrane, an elastomeric membrane, a paper membrane, an extracellularmatrix membrane, a natural membrane or any other suitable membrane. Incertain embodiments, the natural membrane may include collagen, laminin,or a combination thereof. The selection of the pore sizes, materials andother features of the membrane can be varied based on the design of thebiomimetic lung model, the experimental goals, or other suitablemotivations.

In certain embodiments, the layer of cells 30 can be airway epithelialcells. In certain embodiments, the airway epithelial cells can compriseType I and Type II cells. In certain embodiments, the airway epithelialcells can be from all compartments of the lung, including but notlimited to, nasal epithelial cells, tracheal epithelial cells, bronchialepithelial cells, small airway epithelial cells and/or alveolarepithelial cells, (e.g., Type I and II cells). In certain embodiments,the airway epithelial cells are derived from human or animal tissue. Incertain embodiments, the airway epithelial cells can be from healthyhuman or animal lung tissue. In certain embodiments, the airwayepithelial cells can be from diseased human or animal lung tissue (e.g.,fibrosis, emphysema, COPD, bronchitis, asthma, cystic fibrosis). Incertain embodiments, the airway epithelial cells can be differentiatedfrom stem cells (e.g., induced pluripotent stem cells, embryonic stemcells). In certain embodiments, the biomimetic lung model can be a modelfor smoking-induced emphysema or COPD. In certain embodiments, thesmoking-induce emphysema or COPD can involve the pathological structuralchanges in the lung that take place over years. In certain embodiments,the agent causes oxidative stress.

In certain embodiments, the biomimetic lung model contains most of themajor cellular constituents in the airway niches of the human lung. Incertain embodiments, the diseased lung can be chronically diseased. Incertain embodiments, the layer of cells can further comprisemacrophages. In certain embodiments, the macrophages can be alveolar,interstitial, intravascular, airway macrophages and/or an immortalizedcell line (e.g., THP-1). In certain embodiments, the macrophage cellscan be added to the cell layer on the first side of the membrane at aration of about 1 macrophage to about 50 epithelial cells, about 1macrophage to about 45 epithelial cells, about 1 macrophage to about 40epithelial cells, about 1 macrophage to about 35 epithelial cells, about1 macrophage to about 30 epithelial cells, about 1 macrophage to about25 epithelial cells, about 1 macrophage to about 20 epithelial cells,about 1 macrophage to about 18 epithelial cells, about 1 macrophage toabout 16 epithelial cells, about 1 macrophage to about 14 epithelialcells, about 1 macrophage to about 12 epithelial cells, about 1macrophage to about 10 epithelial cells, about 1 macrophage to about 8epithelial cells, about 1 macrophage to about 6 epithelial cells, orabout 1 macrophage to about 5 epithelial cells.

In certain embodiments, the macrophages are added to the gel layer. Incertain embodiments, the macrophages can be added to the gel layer at aratio of about 1 macrophage to about 8 basal stromal cells, about 1macrophage to about 7 basal stromal cells, about 1 macrophage to about 6basal stromal cells, about 1 macrophage to about 5 basal stromal cells,about 1 macrophage to about 4 basal stromal cells, or about 1 macrophageto about 3 basal stromal cells.

In certain embodiments, a second layer of cells 40 can be adhered to thesecond side of the membrane. In certain embodiments, the second layer ofcells can be endothelial cells including pulmonary microvascularendothelial cells. In certain embodiments, the second layer of cells canbe large vessel endothelial cells, arterial endohtelial cells, venousendothelial cells all from lung. In certain embodiments, the secondlayer of cells can be lymphatic endothelial cells. In certainembodiments, the first layer of cells 30 and second layer 40 of cellscan be cultured in apposition on a membrane 20. In certain embodiments,the first or second cell layer can have an artificially inducedpathology. In certain embodiments, the cell layers can be monolayers.

In certain embodiments, a cell-laden gel layer can be added to thebiomimetic model. In certain embodiments, a gel layer can be attached tothe second side of the membrane. In certain embodiments, the gel cancomprise collagen. In certain embodiments, tissue or cells can beembedded in the gel. In certain embodiments, the gel layer allows theembedded cells to communicate with the layer of cells on the first sideof the membrane. In certain embodiments, the cells embedded in the gellayer can be connective tissue or cells. In certain embodiments, thecells embedded in the gel layer can be basal stromal cells. In certainembodiments, the basal stromal cells can be fibroblasts and/orpericytes. In certain embodiments, the cells embedded in the gel layercan be airway and/or vascular smooth muscle cells. In certainembodiments, the cells embedded in the gel layer can be extracellularmatrix proteins. In certain embodiments, the extracellular matrixproteins can be, but not limited to, fibronectin, laminin, hyaluaronicacid and/or similar materials.

Referring to FIG. 3 for the purpose of illustration and not limitation,there is provided an exemplary method for fabricating a biomimetic lungmodel (300). In certain embodiments, the method can include fabricatinga body (301), the body having first and second microchannels disposedthereon. The body, including the microchannels, can be built by anymethods known in the art, including, but not limited to, those outlinedin Huh et al., Nature Protocols 8:2135-2157 (2013).

In certain embodiments, the method can include inserting a membranebetween the first and second microchannels (302) such that the first andsecond microchannels can be in fluid communication through the membrane.In certain embodiments, the membrane can have a first and second side.In certain embodiments, the method can include adhere a layer of cells(303) of a first cell type disposed on a first side of the membrane.

Referring to FIG. 4 for the purpose of illustration and not limitation,there is provided an exemplary method for fabricating a biomimetic lungmodel (400). In certain embodiments, the biomimetic lung model can be amodel of a diseased lung. In certain embodiments, the method can includefabricating a body (401), the body having first and second microchannelsdisposed thereon. In certain embodiments, the method can includeinserting a membrane between the first and second microchannels (402)such that the first and second microchannels can be in fluidcommunication through the membrane. In certain embodiments, the membranecan have a first and second side. In certain embodiments, the method caninclude adhering a layer of cells (303) of a first cell type disposed ona first side of the membrane. In certain embodiments, the method caninclude coupling a device to the body (404). In certain embodiments, themethod can include the device delivering an agent to at least one of themicrochannels.

In certain embodiments, the method can include casting a gel andattaching a gel to the second side of the membrane. In certainembodiments, the casting of a gel can include sulfo-sanpah treatment ofthe membrane material to promote collagen/ECM anchorage. In certainembodiments, the gel is prepared with cells and pipetted onto the secondside of the membrane that has been stamped to a channel. In certainembodiments, the gel is prepared without cells and pipetted onto thesecond side of the membrane. In certain embodiments, after the gel layersolidifies, the upper channel portion—now with a cast gel under themembrane—can be flipped back over and placed over the reservoir layer tocomplete the device assembly.

Referring to FIG. 5 for the purpose of illustration and not limitation,an exemplary method of testing metabolic regulation of lung tissue (500)is provided. In certain embodiments, the method can include providing abiomimetic lung model (501) as disclosed herein, and can includedelivering an agent of interest in one of the first or secondmicrochannels (502). In certain embodiments, the substance of interestcan be, for example, cigarette smoke, nicotine aerosol, automotiveexhausts, dry powder drugs, aerosol drugs, ozone, wood smoke, naturalplant smoke, silica dust, acrylic dust, particulates, asbestos fibers,solvents, grain dust, bird droppings, and animal droppings. In certainembodiments, the method can include simulating physiological flowconditions. In certain embodiments, the method can include simulatingphysiological breathing/inhalation conditions. In certain embodiments,the method can include measuring pathological responses to the agent. Incertain embodiments, the method can include measuring tissue hardeningin response to the agent. In certain embodiments, the method can includemeasuring inflammatory and other abnormal biological responses, forexample, but not limited to, production of cytokines/chemokines &expression of adhesion molecules; activation of oxidative stresspathways, endoplasmic reticulum (protein production) stress; DNA damage;or cell apoptosis and necrosis (death).

In certain embodiments, a device can deliver culture medium to the firstand second microchannels (e.g. FIG. 6 ). In certain embodiments, adevice can deliver culture medium to one of the first or secondmicrochannels. In certain embodiments, a device can deliver culturemedium to only the second microchannel. In certain embodiments, thedevice can pump culture medium to the microchannel(s) through a port(e.g., FIGS. 1 and 2 (15)) in the body, wherein the first opening of theport 15 can be to the outside of the body and the second opening of theport 15 can be to at least one microchannel. In certain embodiments, theculture medium leaves the microchannel through an exit port 16. Incertain embodiments, the device can pump culture medium out of themicrochannel(s) through an exit port 16 in the body, wherein the firstopening of the exit port 16 opens to the microchannel and the secondopening of the exit port 16 can be to the outside of the body. Incertain embodiments the port 15 or exit port 16 only connects to onemicrochannel. In certain embodiments, the pumping system can draw/pullmedium though the channels from a reservoir. In certain embodiments, forthe smoke delivery, the smoke can be pulled through the device. Incertain embodiments, there can be a mixing chamber in the smokegeneration apparatus (i.e., the smoke is generated and diluted/humifiedin a positive pressure flow process, it then fills an open mixingvessel) from which the smoke/agent mixture can be pulled through thedevice at a set flow rate via syringe pump. In certain embodiments, theculture medium is not delivered to the biomimetic model while the agentis being delivered. In certain embodiments, the culture medium isdelivered to one microchannel while the agent is delivered to the othermicrochannel.

In certain embodiments, the device delivers the agent to the firstmicrochannel (e.g. FIG. 7 ). In certain embodiments, the devicedelivering the agent can be an automated machine (e.g. FIG. 8 ). Incertain embodiments, the agent can be delivered to the firstmicrochannel. In certain embodiments, the agent can be more dilute thedeeper it moves into the microchannel. In certain embodiments, the agentcan be delivered at the concentration and intermittent schedule asencountered by a human lung. In certain embodiments, the device candevice can deliver the agent to the microchannel(s) through a port(e.g., FIGS. 1 and 2 (15)) in the body, wherein the first opening of theport 15 can be to the outside of the body and the second opening of theport 15 can be to at least one microchannel.

In certain embodiments, the device delivers smoke (e.g., cigarettesmoke) to the first microchannel. In certain embodiments, the devicedelivers the smoke through the port 15 in the first outer body portion13. In certain embodiments, when the smoke is delivered to the port 15in the first outer body portion 13 it is delivered to only the firstmicrochannel. In certain embodiments, the device delivering the smokecan be an automatic smoking machine (e.g. FIG. 8 ). For example, but notlimited to, the automatic machine could be a benchtop-sized, automatedsmoking machine interfaced with microfluidic devices (e.g., a Human PuffProfile model cigarette smoking machine (CH Technologies)). In certainembodiments, the cigarette smoke can be delivered to the firstmicrochannel such that the distribution of cigarette smoke mimicscigarette smoke exposure conditions experience by the cell lining in thehuman lung. In certain embodiments, the cigarette smoke can be moredilute the deeper it moves through the first microchannel. In certainembodiments, the Weibel model can be used to deliver the smoke. Incertain embodiments, the smoke can be intermittently delivered to modelthe frequency in which a smoker's lungs may experience the smoke (e.g.,to model a heavy versus light smoker). In certain embodiments, theconcentration of the smoke mimics that of a lung exposed to secondhandsmoke.

In certain embodiments, the device can be a model for fibrosis. Incertain embodiments, the fibrosis model entails measuring fibroblastproliferation, fibroblast ECM production, and/or stiffening of the geland/or tissue.

In certain embodiments, the biomimetic lung model can include additionalelements, for example but not limited to, integrated pumps, valves,bubble traps, oxygenators, gas-exchangers, in-line microanalyticalfunctions, and other suitable elements. Such elements can allow foradditional control and experimentation using the biomimetic lung model.In certain embodiments, the biomimetic lung model can include featuresfor automatically performing experiments on the biomimetic lung model.For example, in some embodiment, the biomimetic lung model can includeautomated valve or fluid (e.g., liquid or air) control mechanisms orautomatic testing mechanisms, such as sensors or monitors. In certainembodiments, the biomimetic lung model can be configured to be coupledwith other sensors or monitors not disclosed on the biomimetic lungmodel. In certain embodiments, the biomimetic lung model can include acleaning reservoir coupled to the channels for cleaning or sterilizingthe channels. In certain embodiments, the biomimetic lung model can bemodular in construction, thereby allowing various elements to beattached or unattached as necessary during various cleaning,experimenting, and imaging processes. In certain embodiments, thebiomimetic lung model, or portions thereof, can be reusable, and in someembodiments, the biomimetic lung model, or portions thereof, can bedisposable.

In certain embodiments, the biomimetic lung model compositions disclosedherein can be used to study exchange of various endogenous and exogenoussubstances such as oxygen, nutrients, metabolic waste, and xenobiotics.Furthermore, in certain embodiments, the biomimetic lung model disclosedherein can provide opportunities to develop specialized human diseasemodels that can use patient-derived cells to simulate complexhuman-specific disease processes for a variety of biomedical,pharmaceutical, toxicological, and environmental applications. Forexample, in certain embodiments, the biomimetic lung model disclosedherein can be used to study pulmonary pathologies as well as otherpathophysiologic processes that can occur in the lung. Additionally, incertain embodiments, the biomimetic lung model compositions disclosedherein can be used as a screening tool to evaluate the safety andtoxicity of environmental exposures (e.g., chemicals, toxins) and drugs,and the drug transfer between lung and surrounding tissue.

Cell Culture

In certain embodiments, the layer of cells 30 can be obtained from lungtissue. In certain embodiments, the layer of cells 30 can be obtainedfrom a primary culture generated from lung tissue. Standard techniquesof tissue harvesting and preparation can be used.

In certain embodiments, the layer of cells 30 can be an immortalizedcell line.

In certain embodiments, adhering the layer of cells to the first side ofthe membrane can include standard approaches of extracellular matrixcoating of the membrane, for example, but not limited to the use offibronectin, prior to seeding of cells. In certain embodiments, to seedthe cells, a high density cell suspension can be introduced to thechannel and allowed to incubate under static conditions to allow thecells to adhere. In certain embodiments, the cell suspension is allowedto incubate for 2 to 4 hours. In certain embodiments, after the periodof attachment flow can be initiated to allow the washing away ofunattached cells and beginning the perfused culture stage. In certainembodiments, some cell proliferation can occur to fill out the entiremembrane surface. In certain embodiments, cell proliferation is allowedto occur for 2-3 days.

In certain embodiments, the immune cells are obtained from peripheralblood and incorporated into the lung model. For example, peripheralblood monocytes can be obtained to generate the macrophage cells used inthe model. In certain embodiments, THP-1 cells are used. In certainembodiments, macrophages can be obtained from patient biopsies andbronchoalveolar lavages.

In certain embodiments, the maccrophagees can be introduced into thechannel after the epithelial layer has formed. For example, this can beaccomplished by pipetting them into the channel. As they differentiatethey adhere to the epithelial cells and crawl around. One way to test tosee if they adhered is to wash the channel and check to see if they havenot washed away.

In certain embodiments, the stromal cells are derived from a primarycell culture, established cell culture, or an immortalized cell culture.In certain embodiments, the stromal cells are obtained from a biopsiedtissue.

In certain embodiments, the gel later contains nutrients to feed thecells. In certain embodiments, the cells in the gel layer obtainnutrients from culture medium from the microchannel or reservoir. Incertain embodiments, the cells obtain nutrients from within the geland/or from culture medium from the microchannel or reservoir.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

EXAMPLES Example 1: Smoking-Induced Disease Model of a Human SmallAirway

Cigarette smoking-induced pathology involves induction of cellularstress responses in the epithelial cells lining the airways of humanlungs, including activation of endoplasmic reticulum (ER) stressresponses which result from the cell's inability to cope with itsprotein production demands. Acute smoke exposure causes oxidativestress, a consequence of which is disrupted proteostasis. As an exampleof one such response that can be probed in the biomimetic model, cellshave evolved various mechanisms for coping with disrupted proteostasis,one of which is the Unfolded Protein Response (UPR) (FIG. 9 ). Stresstolerance leads to the return to homeostasis (proteostasis). Failure torestore homeostasis prompts a cell death program. Typically theapoptosis is immunologically silent; however, during heavy stressproinflammatory necrosis is prevalent. Thus, the cells either recover,or they don't and die, which is part of the beginning of the diseaseprocess that leads to COPD, fibrosis or other lung diseases. Any otherdisease relevant signaling pathway or cellular response mechanism can beassayed using standard cell biological methods in addition to the UPR,including but not limited to oxidative stress responses involvingexpression of Nrf-2 and inflammatory responses involving activation ofNFkB coupled with the production and release of inflammation-modulatingcytokines and chemokines.

It has been shown that the UPR is activated in the lungs of smokers withCOPD (Jorgensen et al., 2008; Kelsen et al., 2008) and in the lungs oflaboratory animals after exposure to the smoke of a single cigarette(Kenche et al., 2013), demonstrating the sensitivity of measuring UPRactivation as an early injury response to smoke exposure.

The body of the model was formed using soft lithography techniques, inwhich the PDMS mixture was pour over the mold, and the body was allowedto cure. The microchannels were etched into the body, with thedimensions of 10 mm×1 mm×0.15 mm (length×width×height).

In this example, non-diseased small airway epithelial cells derived fromhealthy human donors (from Lonza) were used. Although Matrigel wasapplied to the membrane prior to seeding of the cells, any ECM coating(e.g. Fibronectin) can be used to enhance initial cell adhesion. A cellsuspension in the range of 2-8 million cells/ml was introduced to thechannel and allowed to incubate under static conditions for 2-4 hours.After the period of attachment, flow was initiated to wash awayunattached cells. After cell proliferation was allowed to occur for 1-3days, the medium was removed to initiate air-liquid interface culture.The length of submerged culture varies depending on the density ofinitial seeding.

The device delivering the cell culture medium to the microchannels wasdisconnected from the body of the model before smoke was delivered tothe microchannel above the membrane. Cell culture media remained in thelower microchannel to nourish the cells. A picture of the membranepopulated by human small airway epithelial cells in air-liquid interfaceculture is shown in FIG. 10 .

A lit cigarette was placed into a chamber to allow the smoke toaccumulate. The air with smoke was channeled over the cells by pullingthe smoke from the chamber through the upper microchannel of the modelvia a syringe device attached to the body of the model via a connectingtube.

UPR activation was measured by examining small airway epithelialexpression of ATF6 and the phosphorylated form of EIF2a viaimmunohistochemistry and fluorescence microscopy.

Up-regulation and nuclear translocation of ATF6 was observed (FIG. 11 ).Phosphorylation of EIF2a was also observed (FIG. 12 ) following exposureto highly diluted smoke of a single cigarette for approximately 2-3minutes, demonstrating a highly sensitive cellular readout of earlyinjury in response to smoke exposure.

Even after exposure to small amounts of cigarette smoke (fractions ofindividual puffs), an increase in UPR protein staining (AFT6 green,pEIF2a red) was induced (FIG. 14B).

After 4 hours of smoke exposure at a dilution ration of 1-10% anincrease in cellular injury was observed (FIG. 14A) as compared to cellsexposed only to air (FIG. 14B).

After 12 hours of smoke exposure at a dilution ration of 1-10% there wasa dramatic change in the cellular morphology of the airway epithelialcells. In particular, a greater percentage of the cells were rounded,which indicated that the cells were likely dying by one of multiplemechanisms including necrosis or apoptosis (FIG. 15 ).

After 16 hours, very low levels of UPR activation (i.e., stressresponse) is seen in the control, air treated, cells (FIG. 16A). On theother hand, after 16 hours of exposure to smoke there was robust UPRactivation in the exposed bronchial epithelial cells (FIG. 16B).

Single smoke exposure induced acute injury of human bronchial epithelialcells and small airway epithelial cells, leading to significant loss ofepithelial integrity and barrier function. This injurious response wasaccompanied by increased stress in the endoplasmic reticulum, asmanifested by robust activation of the unfolded protein response.

FIGS. 17A-B depict simulation data of lung models subject to cigarettesmoke particles (FIG. 17A) and acrolein vapor (FIG. 17B). The disclosedsimulation data indicates that dilutions of ˜95% or greater are requiredto replicate the amounts of exposure expected at the depth of the smallairways in a human smoker. These data in part informed the initial rangeof concentrations tested and the responses of cells in our biomimeticmodel corroborate these predictions. Particles less than 1% at entranceto small airways. Acrolein <4-5% at entrance to small airways.

FIGS. 18A-F depict morphology and viability results of small airwayepithelial cells exposed to smoke of a single cigarette. FIGS. 18A, 18C,and 18E depict morphology of small airway epithelial cells exposed tosmoke of a single cigarette. FIGS. 18B, 18D, and 18F depict viabilityresults of small airway epithelial cells exposed to smoke of a singlecigarette corresponding to FIGS. 18A, 18C, and 18E, respectively. FIGS.18A and 18B depict epithelial cells exposed to smoke of a singlecigarette at 86% dilution. FIGS. 18C and 18D depict epithelial cellsexposed to smoke of a single cigarette at 93% dilution. FIGS. 18E and18F depict epithelial cells exposed to smoke of a single cigarette at96.5% dilution under stable ALI post-exposure. As illustrated by FIGS.18A-F, the integrity of the epithelial barrier maintained only at thehigh dilution of 96.5%. This is a requirement for performing multiplesmoke exposures, and at the same time demonstrates the sensitivity ofSAEC in the biomimetic model to small amounts of smoke. These resultsare on par with computer simulation predictions of expected smokeconstituent concentrations at the level of the small airways asillustrated by FIG. 19 .

FIGS. 19A-C depict reduction of UPR activation following initial smokeexposure. FIGS. 19A, 19B, and 19C illustrate recovery and/or homeostasisresults at 16 hours, 40 hours, and 64 hours, respectively. Asillustrated by FIGS. 19A, 19B, and 19C, maintenance of the tissue layerintegrity at low smoke dilution can allow for continuation of theexperiments without channel filling to observe recovery of the cellsfrom the initial injury. Additionally, maintenance of the tissue layerintegrity at low smoke dilution can facilitate subsequent smokeexposures, such as the 8 day regimen used in the demonstrations offibrotic response to smoke exposure of an incorporated stromal gellayer.

Example 2: COPD Disease Model

A biomimetic lung model was fabricated to mimic COPD in small airwaycells. This model can be used to study modulation of the dysfunctionalstate in the epithelial cells, and to potentially discover/develop newtherapeutics. Furthermore, outputs established using COPD-derived cellsset a standard for establishing disease-relevant phenotypes in normal,healthy cells exposed to cigarette smoke as described in Example 1.

The body of the model was formed using soft lithography techniques, inwhich the PDMS mixture was pour over the mold, and the body was allowedto cure. The microchannels were etched into the body, with thedimensions of 10 mm×1 mm×0.15 mm (length×width×height).

Cells isolated from the lungs of smokers with COPD small airway cellswere obtained from Lonza. A 2-8 million cells/ml density cell suspensionwas introduced to the channel and allowed to incubate under staticconditions for 2-4 hours. After the period of attachment, flow wasinitiated to wash away unattached cells. After cell proliferation wasallowed to occur for 1-3 days, the medium was removed to initiateair-liquid interface culture.

A lit cigarette was placed into a chamber to allow the smoke toaccumulate. The air with smoke was channeled over the cells by pullingthe smoke from the chamber through the upper microchannel of the modelvia a syringe device attached to the body of the model via a connectingtube.

Cells were stained for expression of ATF6 and pEIF2a, which are markersof the UPR response. They show high levels of activation in allconditions, which is indicative of their pathology. When smoked wasdelivered to the regular/normal airway cells, they started to expressthese same proteins found constitutively in the COPD cells (shown inExample 1).

The cells were examined by immunohistochemistry and fluorescencemicroscopy. The similarity of the staining in both the control and thesmoke exposed COPD cells demonstrated that the COPD cells have thedisease characteristics regardless of in vitro smoke exposure (FIG. 20).

Example 3: Biomimetic Lung Model with Basal Stromal Tissue and AirwayLumen Macrophages

A biomimetic lung model was fabricated to include both basal stromaltissue and airway lumen macrophages (FIG. 21 ).

The body of the model was formed using soft lithography techniques, inwhich the PDMS mixture was pour over the mold, and the body was allowedto cure. The microchannels were etched into the body, with thedimensions of 10 mm×1 mm×0.15 mm (length×width×height).

In this example, non-diseased small airway epithelial cells from healthyhuman donors (Lonza) were used. A 2-8 million cells/ml density cellsuspension was introduced to the channel and allowed to incubate understatic conditions for 2-4 hours. After the period of attachment, flowwas initiated to wash away unattached cells. After cell proliferationwas allowed to occur for 1-3 days, the medium was removed to initiateair-liquid interface culture.

The gel can be created by adding 1-8 mg of collagen to water, dependingon the desired gel density and/or stiffness, and the liquid gel was keptat 4° C. In these examples, 2 mg/ml collagen gels were used. Instanceswhen cells were added to the gel, they were added during this liquidphase. The membrane was treated with sulfo-sanpah to promotecollagen/ECM anchorage. The gel was pipetted onto the underside of amembrane that had been stamped to the microchannel while the device wasflipped upside down. Once the gel layer solidified by incubating at 37°C., the upper channel portion—now with a cast gel under the membrane—wasflipped back over and placed over the reservoir layer to complete thedevice assembly.

The epithelial cells remained viable once the gel layer was attached tothe underside of the membrane. In particular, FIG. 22 shows that after72 hours after the attachment of the gel layer, the epithelial cells andstromal cells (fibroblasts) in the air-liquid interface configurationremained viable. Viability studies were conducted with an live/deadstain (calcein-AM and ethidium bromide) for simultaneous fluorescencestaining of viable and dead cells.

A THP-1 monocyte/macrophage cell line was also seeded onto the bronchialepithelial cell-lined channel. Staining with cell tracker die indicatedthat adherent/crawling macrophage-like cells were present on the surfaceof the airway epithelium, mimicking the multicellular complexity of thein vivo airway niche (FIG. 23 ).

Example 4: Fibrosis Disease Model

A biomimetic lung model is fabricated to mimic fibrosis in the smallairway tissue niche of the human lung. This model can be used to studyhow modulation of the dysfunctional state in smoked epithelial cells caninfluence the fibroblasts and/or stromal cells in the adjacent gel layerof the biomimetic model, and to potentially discover/develop newtherapeutics that inhibit pathological processes and promote tissuehomeostasis.

In this model, the epithelial cells can be seeded onto the first side ofthe membrane within the upper microchannel. Fibroblasts are added to thegel layer prior to being cast and set upon the second side of themembrane as described above.

The present disclosure is well adapted to attain the ends and advantagesmentioned as well as those that are inherent therein. The particularembodiments disclosed above are illustrative only, as the presentdisclosure can be modified and practiced in different but equivalentmanners apparent to those skilled in the art having the benefit of theteachings herein. Furthermore, no limitations are intended to thedetails of construction or design herein shown, other than as describedin the claims below. It is therefore evident that the particularillustrative embodiments disclosed above can be altered or modified andall such variations are considered within the scope and spirit of thepresent disclosure. Various publications, patents and patent applicationare cited herein, the contents of which are hereby incorporated byreference in their entireties.

The invention claimed is:
 1. A biomimetic lung model comprising: a bodyhaving a first microchannel and a second microchannel disposed therein;a membrane disposed between the first microchannel and the secondmicrochannel, the membrane having a first side facing the firstmicrochannel and a second side facing the second microchannel; a layerof cells coating the first side of the membrane; and tissue or cellsembedded within a gel layer attached to the second side of the membrane,the embedded tissue or cells being in communication with the layer ofcells on the first side of the membrane, wherein at least one agent isdelivered through at least one of the first microchannel and the secondmicrochannel, and the layer of cells comprises airway epithelial cellsincluding one or more of nasal epithelial cells, tracheal epithelialcells, and small airway epithelial cells.
 2. The biomimetic lung modelof claim 1, wherein the membrane comprises one or more of polyester thinclear fabric, polydimethylsiloxane, polymeric compounds, and naturalmembranes.
 3. The biomimetic lung model of claim 2, wherein the naturalmembranes comprising at least one of collagen and laminin.
 4. Thebiomimetic lung model of claim 1, wherein the first microchannel has awidth from about 0.5 mm to about 2 mm and a length from about 1000 μm toabout 10 mm.
 5. The biomimetic lung model of claim 4, wherein the firstmicrochannel has a width from about 1 mm to about 2 mm and a length ofabout 1000 μm.
 6. The biomimetic lung model of claim 1, wherein thelayer of cells further comprises at least one type of macrophage cells,the at least one type of macrophage cells being one or more of alveolar,interstitial, intravascular, airway macrophages, and an immortalizedmacrophage cell line.
 7. The biomimetic lung model of claim 1, whereinthe at least one agent comprises one or more of cigarette smoke,nicotine aerosol, wood smoke, natural plant smoke, silica dust, acrylicdust, particulates, asbestos fibers, solvents, grain dust, birddroppings, and animal droppings.
 8. The biomimetic lung model of claim7, wherein the cigarette smoke is delivered to the first microchannelsuch that the distribution of cigarette smoke mimics cigarette smokeexposure conditions experienced by cell linings in the human lung. 9.The biomimetic lung model of claim 1, wherein the gel layer comprisesextracellular matrix proteins, the extracellular matrix proteins beingat least one of collagen, fibronectin, laminin, and hyaluronic acid. 10.The biomimetic lung model of claim 1, wherein the embedded tissue orcells comprise basal stromal tissue or cells.
 11. The biomimetic lungmodel of claim 1, wherein the airway epithelial cells are obtained froma healthy human lung or a chronically diseased human lung.
 12. Thebiomimetic lung model of claim 1, wherein the biomimetic lung model isconfigured to identify pharmaceutical compositions that alleviate a lungdisease.
 13. The biomimetic lung model of claim 1, wherein thebiomimetic lung model is configured to identify agents harmful to thelung.
 14. The biomimetic lung model of claim 1, wherein the at least oneagent comprises cigarette smoke or nicotine aerosol, and the at leastone agent is delivered to at least one of the first microchannel and thesecond microchannel of the biomimetic lung model at a dilution ratio ofbetween about 1% agent and about 10% agent.
 15. A method for fabricatinga biomimetic lung disease model comprising: (a) fabricating a bodyhaving a first microchannel and a second microchannel disposed thereon;(b) inserting a membrane between the first microchannel and the secondmicrochannel, the membrane having a first side facing the firstmicrochannel and a second side facing the second microchannel; (c)adhering airway epithelial cells on a surface of the first side of themembrane; (d) casting a cell- or tissue-laden gel on a surface of thesecond side of the membrane, the gel being configured to solidify byincubation at body temperature; and (e) delivering an agent to at leastone of the first microchannel and the second microchannel, wherein theagent is at least one of cigarette smoke, nicotine aerosol, wood smoke,natural plant smoke, silica dust, acrylic dust, particulates, asbestosfibers, solvents, grain dust, bird droppings, and animal droppings, andthe airway epithelial cells adhered on the surface of the first side ofthe membrane includes one or more of nasal epithelial cells, trachealepithelial cells, and small airway epithelial cells.
 16. The method ofclaim 15, wherein the adhering the airway epithelial cells comprisesseeding a high density cell suspension upon the first side of themembrane and incubating the cell suspension for about 2 hours to about 4hours.
 17. The method of claim 15, wherein the cell- or tissue-laden gelis laden with connective tissue.
 18. The method of claim 15, wherein thecigarette smoke is delivered to the first microchannel and culture mediais delivered to the second microchannel.
 19. The method of claim 15,further comprising integrating macrophage cells among the airwayepithelial cells.